A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages

J Immunol Methods. 1983 Feb 25;57(1-3):373-9. doi: 10.1016/0022-1759(83)90097-2.

Abstract

A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red blood cells fluoresce at about 585 nm when excited at 490 nm and that when trypan blue is in intimate contact with fixed red blood cells, the fluorescence is converted from a chartreuse to a red color. Thus, the ingestion of fixed erythrocytes by murine macrophages can be monitored by fluorescence microscopy after the addition of 1 mg/ml of trypan blue at the end of the assay. Extracellular glutaraldehyde-fixed red blood cells fluoresce a red color whereas the intracellular particles continue to fluoresce a chartreuse color. This method offers a simple and convenient technique for rapidly distinguishing between intracellular and extracellular glutaraldehyde-fixed red blood cells in macrophages.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Erythrocytes / physiology
  • Fluorescent Dyes
  • Glutaral
  • Macrophages / immunology*
  • Mice
  • Phagocytosis
  • Spectrometry, Fluorescence
  • Trypan Blue

Substances

  • Fluorescent Dyes
  • Trypan Blue
  • Glutaral